Immunological methods. Diagnosis of viral infections.
Immunological methods rely on the specific interaction between antigens and antibodies. These methods are central to both research and the diagnosis of viral infections. They help detect either viral antigens or the antibodies produced by the body in response to viral infections. The antigen-antibody complex formed in such reactions can be observed and measured using a variety of laboratory techniques.
Basic Principles
- Immunological tests are based on the specific binding between an antigen and its corresponding antibody.
- These tests may provide qualitative results, simply confirming the presence or absence of a reaction, or quantitative results, determining the concentration of the antigen or antibody.
- To detect an antigen, the test uses specific antibodies. To detect an antibody, the test uses a known antigen.
- The complex formed by the binding of antigen and antibody can be visualized using various techniques depending on the type of test.
Major Immunological Methods
1. Agglutination Tests
- Definition: Agglutination refers to the clumping of cells or particles when antibodies bind to antigens on their surface.
- Mechanism: Antibodies with multiple binding sites (especially Immunoglobulin M) cross-link multiple antigen-bearing cells, forming visible clumps.
- Qualitative Agglutination: Indicates the presence of both antigen and antibody. The clumping is visible to the naked eye or under a microscope.
- Quantitative Agglutination (Antibody Titration):
- Measures the concentration of specific antibodies.
- Involves preparing serial dilutions of a serum sample.
- The highest dilution that still produces a visible agglutination is referred to as the titer.
2. Precipitation Tests
- Used when the antigen is soluble rather than cell-bound.
- Precipitation occurs when antigen and antibody are present in optimal proportions.
- Ring Precipitation Test: An older method where a visible ring forms at the junction of the antigen and antibody layers in a test tube.
- Double Immunodiffusion (Ouchterlony Test):
- Conducted in agar gel.
- Both antigen and antibody diffuse through the gel and form a visible precipitate band when they meet.
- Useful for comparing antigenic similarity and estimating relative antigen concentrations.
3. Immunocytochemistry and Western Blot (Immunoblot)
- Immunocytochemistry:
- Detects antibodies within cells or tissues using fluorescently labeled secondary antibodies.
- Applied for identifying autoantibodies (e.g., antinuclear antibodies in autoimmune diseases).
- Western Blot:
- Detects specific antibodies in patient sera against known viral proteins.
- Involves separating viral proteins via electrophoresis, transferring them to a membrane, and probing with patient serum followed by labeled secondary antibodies.
- Widely used for diagnosing infections like Lyme disease and HIV.